This invention concerns pharmaceutical compositions that are useful for inhibiting formation of amyloid deposits in amyloidogenic disorders, such as those deposits associated with protease resistant prion proteins in transmissible spongiform encephalopathies.
Amyloid formation is found in a number of disorders, such as diabetes, Alzheimer""s Disease (AD), scrapie, Gerstmann-Staussler-Scheinker (GSS) Syndrome, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD), chronic wasting disease (CWD), and related transmissible spongiform encephalopathies (TSEs). These and other diseases in which amyloid plaques or amyloid deposits are formed in the body are referred to as amyloidogenic diseases.
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that include such human disorders as CJD, kuru, fatal familial insomnia, and GSS. Animal forms of TSE include scrapie in sheep, CWD in deer and elk, and bovine spongiform encephalopathy in cattle. These diseases are characterized by the formation and accumulation in the brain of an abnormal proteinase K resistant isoform (PrP-res) of a normal protease-sensitive host-encoded prion protein (PrP-sen). PrP-res is formed from PrP-sen by a post-translational process involving conformational changes that convert the PrP-sen into a PrP-res molecular aggregate having a higher xcex2-sheet content. The formation of these macromolecular aggregates of PrP-res is closely associated with TSE-mediated brain pathology in which amyloid deposits of PrP-res are formed in the brain, which eventually becomes xe2x80x9cspongiformxe2x80x9d (filled with holes).
In the past, the TSE diseases were a medical curiosity because the transmissible agent was difficult to inactivate with heat, radiation or chemicals that would be expected to inactivate infectious living organisms such as bacteria and viruses. Instead, this class of diseases appeared to be transmitted by exposure to an unusual agent, for example by ritual cannibalism in the Foret people of New Guinea, or feeding of animal parts to cattle in bovine spongiform encephalopathy (BSE), latrogenic CJD has also been caused by administration of human growth hormone derived from cadaveric pituitaries, transplanted dura mater and corneal grafts, as well as exposure of surgeons to affected tissue during neurological procedures. The TSE diseases took on new urgency, however, when it appeared that cross-species infection of humans in Europe may have occurred, perhaps from the ingestion of beef from affected cows. That development has further stimulated an international search for a better understanding of the pathophysiological mechanism of the disease, and possible treatments.
The presence of a native prion protein (PrP) has been shown to be essential to pathogenesis of TSE. The cellular protein PrP-sen is a sialoglycoprotein encoded by a gene that in humans is located on chromosome 20. The PrP gene is expressed in neural and non-neural tissues, with the highest concentration of its mRNA being in neurons. The translation product of the PrP gene consists of 253 amino acids in humans, 254 in hamsters and mice, 264 amino acids in cows, and 256 amino acids in sheep (all of these sequences are disclosed in U.S. Pat. No. 5,565,186, which describes methods of making transgenic mice that express species specific PrP). In prion protein related encephalopathies, the cellular PrP-sen is converted into the altered PrP-res that is distinguishable from PrP-sen in that PrP-res aggregates (Caughey and Chesebro, 1997, Trends Cell Biol. 7, 56-62); is proteinase K resistant in that only approximately the N-terminal 67 amino acids are removed by proteinase K digestion under conditions in which PrP-sen is completely degraded (Prusiner et al., 1996, Sem. Virol. 7, 159-173); and has an alteration in protein conformation in which the amount of xcex1-helical conformation for PrP-sen is reduced, and the amount of xcex2-sheet conformation for PrP-res is increased (Pan et al., 1993, Proc. Natl. Acad. Sci. USA 90, 10962-10966).
If PrP-sen is not expressed in the brain tissue of animal recipients of scrapie-infected neurografts, no pathology occurs outside the graft, demonstrating that PrP-res and PrP-sen are both required for the pathology (Brander et al., Nature 379:339-343, 1996). The long latency period between infection and the appearance of disease (months to decades depending on species) has prompted the development of a cell-free in vitro test, in which PrP-res induces the conversion of PrP-sen to PrP-res (Kocisko et al., Nature 370:471474, 1994). See also Prusiner et al., WO 97/16728 published May 9, 1997. The in vitro interaction between PrP-res and PrP-sen occurs with species and strain specificities that mimic TSE species barrier effects and strain differences in vivo (Kocisko et al., 1995, Proc Natl Acad Sci USA 92, 3923-3927; Bessen et al., 1995, Nature 375 , 698-700; Bossers et al., 1997, Proc. Natl. Acad. Sci. USA 94, 49314936; Raymond et al., 1997, Nature 388, 285-288), hence in vitro cell free culture techniques are considered to accurately predict pathological developments in the brains of infected animals. These in vivo and in vitro observations indicate that direct interactions between PrP-res and PrP-sen form PrP-res and promote TSE pathogenesis.
Small synthetic peptides containing certain PrP sequences have previously been shown to spontaneously aggregate to form fibrils with a high degree of xcex2-sheet secondary structure of the type seen in the insoluble deposits in TSE afflicted brains (Gasset et al. , 1992, Proc. Natl. Acad. Sci. USA 89, 10940-10944; Come et al., 1993, Proc. Natl. Acad. Sci. USA 90, 5959-5963; Forloni et al., 1993, Nature 362, 543-546; Hope et al., 1996, Neurodegeneration 5, 1-11). Moreover, other synthetic PrP peptides have been shown to interact with PrP-sen molecules to form an aggregated complex with increased protease-resistance (Kaneko et al., Proc. Natl. Acad. Sci. USA 92, 11160-11164, 1995; Kaneko et al., J. Mol. Biol. 270, 574-586, 1997).
The bovine spongiform encephalopathy epidemic and the appearance of the new variant of CJD in humans has heightened the urgency to develop therapies for the transmissible spongiform encephalopathies (TSE) or prion diseases. U.S. Pat. No. 5,134,121 disclosed the use of a nerve growth blocking peptide to treat prion associated diseases by inhibiting PrP-res formation in the infected host. Sulfated glycans and the sulfonated amyloid stain Congo Red are known inhibitors of PrP-res formation and scrapie agent replication in scrapie-infected neuroblastoma (ScNB) cells (Caughey and Chesebro, 1997, Trends Cell Biol. 7, 56-62; Caughey et al., 1992, J. Neurochem. 59, 768-771; Caughey et al., 1993, J. Virol. 67, 643-650; Caughey et al., 1993, J. Virol. 67, 6270-6272). These polyanions are also protective against scrapie in rodents if administered near the time of infection, but have less therapeutic benefit after the infection has reached the central nervous system (Ehlers and Diringer, 1984, J. Gen. Virol. 65, 1325-1330; Farquhar and Dickinson, 1986, J. Gen. Virol. 67, 463-473; Kimberlin and Walker, 1986, Antimicrob. Agents Chemother. 30, 409-413; Ingrosso et al., 1995, J. Virol. 69, 506-508). This problem, and/or inherent toxicity, also limit the utility of other classes of potential drugs, such as the polyene antibiotics (Demaimay et al., 1997 J. Virol. 71, 9685-9689) and anthracycline (Tagliavini et al., 1998, Science 276, 1119-1122).
Protohemin has been reported to inhibit a protein kinase that phosphorylates filament proteins in patients with Alzheimer"" disease. (Vincent and Davies, 1992, PNAS USA 89:2878-82). Porphyrins and other tetrapyrroles have previously been used therapeutically against cancers and against viral infections (Schuitmaker et al., 1996, J. Photobiol. 34 (1): 3-12; Petho, 1995, Acta Physiol. Hung. 83(2): 113-119). A major therapeutic use has exploited the light absorbing qualities of these molecules to perform xe2x80x9cphotodynamic therapyxe2x80x9d (PDT). In PDT, tissue is photosensitized with the porphyrin or other tetrapyrrole, by exposure to light of a particular wavelength. Absorption of light radiation causes destruction of the sensitized cells (Rebeiz et al., 1996, Cancer Res. 56(2): 399-344; Gomer et al., 1996, J. Clin. Laser Med. Surg. 14(5): 315-321; Fritsch et al., 1998, Arch Dermatol 134(2): 207-214). Alternatively, compounds such as aminolevulinic acid (ALA) may be used to promote the endogenous production of protoporphyrins, followed by photic stimulation and cell death (Schuitmaker et al., 1996, J. Photobiol. 34 (1): 3-12).
There is a need for agents that will specifically inhibit the formation of PrP-res from PrP-sen, and therefore prevent or slow the deposition of amyloid deposits in the tissues of animals that have been exposed to a TSE etiological agent, or are suffering from a neurodegenerative disorder having the characteristics of a spongiform encephalopathy. There also is a need for an agent that has a high inhibitory activity against PrP-res formation, that crosses the blood-brain barrier, and that has only minimum cytotoxic effect, without a detrimental effect on the rate of natural PrP-sen biosynthesis.
The present invention includes compositions and methods for preventing, or inhibiting, the progression of amyloidogenic diseases such as prion associated diseases in which PrP-sen is converted to PrP-res. The compositions of the invention include a pharmaceutically acceptable carrier and a therapeutically effective amount of a tetrapyrrole that inhibits progression of the amyloidogenic disease. In particular embodiments, the tetrapyrrole inhibits the conversion of protease sensitive prion protein (PrP-sen) to protease resistant prion protein (PrP-res).
Particularly disclosed tetrapyrroles include porphines, porphyrins, phthalocyanines, their anionic derivatives (such as sulfonated, carboxylated, and phosphorylated derivatives), their cationic derivatives (such as quaternized or protonated amines), and dervatives bearing uncharged polar groups such as hydroxyl. Particular examples of these tetrapyrroles include phthalocyanine sulfonates, deuteroporphyrins, and meso-substituted porphines. Some particularly disclosed embodiments are relatively stable and non-toxic (having low dark toxicity), and do not include protohemin or protohematin. The intrinsic lipophilicity of the tetrapyrroles makes them more likely to be delivered through the blood brain barrier than many other drugs, which is important in a disease such as TSE in which the most devastating effect of the disease is in the central nervous system. However, the therapeutic tetrapyrroles of the present invention can be delivered peripherally (for example via systemic administration to the lymphoreticular system, including the tonsils and spleen) without toxicity at reasonably high doses.
The invention also includes methods for treating or preventing progression of an amyloidogenic disease in an animal, by administering a therapeutically effective amount of a tetrapyrrole to the animal. In particular embodiments, the amyloidogenic disease is a disease in which inhibiting conversion of PrP-sen to PrP-res in an animal inhibits or prevents progression of the disease, such as a transmissible spongiform encephalopathy (TSE). In other embodiments, the disease is one involving pathological formation of amyloid depostis or plaques, such as Alzheimerxe2x96xa1s disease or Type 2 diabetes.
In embodiments of the invention in which the tetrapyrrole is a phthalocyanine, the phthalocyanine may be a phthalocyanine sulfonate, such as a phthalocyanine monosulfonate, disulfonate, trisulfonate, or tetrasulfonate, and/or a metallophthalocyanine with a metal compound, such as VO, or a metal ion, such as a cation of iron, manganese, cobalt, nickel, zinc, and aluminum, occupying the center of the macrocyclic ring. In particular examples the metal compound or metal ion may be Fe3+, Mn3+, Co3+, Cu2+, Ni2+, Zn2+, Co2+, Al3+, or VO, and especially the subgroup Fe3+, Mn3+, Co3+, Cu2+, Ni2+, and Zn2+. In embodiments in which the tetrapyrrole is a tetrasulfonylphthalocyanine (PcTS), the particular PcTS includes metal-free PcTS, PcTS-Fe3+, PcTS-Mn3+, PcTS-Co3+, PcTS-Cu2+, PcTS-Ni2+ and PcTS-VO.
In embodiments in which the tetrapyrrole is a deuteroporphyrin, the deuteroporphyrin may be a derivative substituted with, for example, a sulfato, a carboxylate, an acetyl, an oxime, a ketone, halogen or nitrate group, or derivatives thereof, or a hydroxyalkyl or polyhydroxyalkyl group, having for example, up to five carbons, and particularly three or fewer carbons (such as hydroxymethyl, hydroxyethyl, hydroxy propyl, dihydroxyethyl, and dihydroxypropyl). In particular embodiments, the derivative may be a sulfonate. For embodiments in which the deuteroporphyrin is complexed with a metal, the metal ion may be an iron ion, for example Fe3+.
In those embodiments in which the tetrapyrrole is a meso-substituted porphine, the meso-porphine may, for example, be substituted at one or more of the meso postitions with alkyl, phenyl, carboxyl, 2-pyridyl, 3-pyridyl, or 4-pyridyl groups, or combinations thereof. Phenyl groups at the meso positions may further be substituted with a carboxylate group,a sulfate group, secondary, tertiary, and quaternary amine groups, hydroxyl groups, or phosphate groups. Pyridyl groups at the meso positions may further be N-substituted with alkyl groups to form quaternary pyridinium ions. Alternatively, the basic pyridyl groups can be protonated at appropriate pH""s to form cationic pyridinium groups. The meso-porphine may be a metalloporphine that is complexed with a metal compound (such as VO) or a metal ion selected from the group of iron, manganese, copper, nickel and zinc, ions and particularly a cation selected from the group of: Fe3+, Mn3+, Co3+, Cu2+, Ni2+, Zn2+, Co2+ and Al3+.
When administered as a therapeutic treatment, the tetrapyrrole may be administered at a dose between 1 nanogram and 0.5 to 1 gram per kg body weight (for example 1 microgram to 100 micrograms per kg) of the animal, without appreciable toxicity, such as cytotoxicity. Although the tetrapyrrole may be administered by a wide variety of routes (direct administration into the CNS, intracranial ventricular, intrathecal, aural, transdermal, intravenous, intramuscular, subcutaneous, oral, olfactory, ocular and rectal), the tetrapyrroles of the present invention are particularly advantageous because many of them are believed to readily pass the blood brain barrier. Compounds having increased lipophilicity, or in pharmaceutical carriers such as liposomes, will have enhanced penetration of the CNS.
The invention also includes methods of screening compounds which inhibit conversion of PrP-sen to PrP-res, by contacting PrP-sen with a tetrapyrrole or an analog or derivative or mimetic thereof, in a mixture of PrP-res and PrP-sen under conditions in which a conversion of PrP-sen to PrP-res would be expected to occur. The mixture is exposed to a sufficient concentration of proteinase K to proteolytically degrade PrP-sen after exposure of the proteinase K, and then an assay is performed to determine whether the conversion of PrP-sen to PrP-res has been inhibited. One such method for detecting this inhibition is by detecting the relative or complete disappearance of an electrophoretic band that corresponds to PrP-res, as an indication that the peptide inhibits conversion of PrP-sen to PrP-res.